During the initial funding period, our group developed a novel Diffusion Basis Spectrum Imaging (DBSI) method to simultaneously detect and quantitate inflammation, demyelination and axon injury in vivo using diffusion imaging (Wang et al. 2011). DBSI has substantially improved the accuracy and specificity of our prior diffusion tensor imaging (DTI) approach, by overcoming the main inadequacies of DTI. We hypothesize that DBSI can quantitate the proportions of axon injury, demyelination, and inflammation in CNS of MS patients. We previously showed using a mouse model that DBSI detects and quantitates axonal and myelin injuries that had escaped detection by standard imaging, and by DTI. Our preliminary data now include validation of DBSI using autopsied and biopsied human specimens, with favorable correlations with human histology. We also now have longitudinal data spanning 1.5 yrs, and comparisons of DBSI with magnetization transfer imaging (MTI). In Project 3, we will apply DBSI to humans with MS, comparing it to standard MRI, DTI and MTI. Project 3 will classify MS lesion subtypes by measures of axon injury, demyelination, and inflammation (cellularity and increased free water due to edema or tissue loss), and follow the patients over 4 years to identify predictors and correlates of clinical deterioration. We expect to achieve this using DBSI by differentiating prominent axonal injury vs. axon preservation, and demyelination vs. myelin preservation/ remyelination. We will examine established persistent black holes (PBHs) (new sub-aim), and perform longitudinal assessments of gadolinium-enhancing (Gd+) MS lesions to determine if DBSI will predict PBH formation, representing severe axon loss. With its ability to profile lesions and normal-appearing CNS, DBSI could help non-invasively elucidate the substrate of MS lesion formation and detect inflammation behind an intact blood-CNS-barrier (not detected by Gd+). DBSI has potential to aid development and testing of new therapies for progressive MS where loss of axons and tissue integrity are believed to play a large role.